Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis.

By using oligonucleotide-directed mutagenesis, Lys-112 of bovine growth hormone (bGH) was changed to leucine, and its resulting effect on folding was studied. Equilibrium denaturation curves for the mutant protein exhibit biphasic or nonsymmetrical transitions by a variety of spectroscopic and hydrodynamic techniques, whereas the wild-type protein at the same concentration exhibits symmetrical transitions. The mutant protein refolds slower (by a factor of 30) and more readily precipitates upon refolding than the wild-type protein. These folding characteristics of the mutant protein are demonstrated to be a result of stabilization of an associated folding intermediate. A 38-amino acid fragment (96-133) derived from the mutant protein is helical, likely amphipathic, and more stabilized by increasing peptide concentration than is the corresponding helical peptide from the wild-type protein. The increased stability of the associated intermediate and the increased helicity of the peptide from the mutant protein are explained by preferential intermolecular interactions between helices due to enhanced hydrophobic attraction by their amphipathic surfaces.